Thermosensitive hydrogels for 3D bioprinting of cartilage constructs

Tissue engineering (TE) aims to regenerate damaged tissues by the combined use of biomaterials and cells, often in presence of bioactive molecules, such as growth factors. Particularly for tissues with poor regenerative capacity, such as articular cartilage, TE approaches may lead to promising treatments. Articular cartilage is a connective tissue responsible for absorbing and distributing the load acting on the joint, to the undelaying bone. The limited regenerative capacity of articular cartilage is due to the absence of blood vessels in this tissue and consequent low cellular activity. Currently, an optimal treatment for cartilage defects does not exist. Therefore, TE strategies are exploited to obtain cell-laden scaffolds able to provide an initial mechanical support that over time degrades, while new tissue is formed by the embedded cells. The aim of this thesis was to develop biomechanically advanced hydrogel constructs for articular cartilage regeneration.
Chapter 1 provides a general introduction of the anatomy and the physiology of articular cartilage, as well as the current clinically relevant techniques for its repair. Considering the limitations of present treatments, TE strategies for cartilage regeneration are presented as a promising alternative. Moreover, three-dimensional (3D) bioprinting is presented as a novel and versatile technology for the accurate design and generation of cartilage constructs. The experimental “core” of this thesis describes a step-by-step development of thermosensitive and photo-crosslinkable hydrogels based on partially methacrylated poly[N-(2-hydroxypropyl)methacrylamide mono-dilactate]/polyethylene glycol triblock copolymers (pHPMAlac-PEG) and chemically modified polysaccharides, i.e. methacrylated chondroitin sulfate (CSMA) or hyaluronic acid (HAMA), for 3D bioprinting of cartilage constructs.
In Chapter 2, 3 and 4 an extensive and comprehensive in vitro characterization of this material is reported. The synthesis of the polymeric building blocks is achieved with a full control over polymer characteristics. Further, these building blocks are used to fabricate hydrogels with favorable rheological behavior for 3D bioprinting application and able to support cartilage-like tissue formation by embedded chondrocytes. Chapter 5 describes an introductory study on the possibility to enrich pHPMAlac-PEG hydrogels with protein-laden microgels for a controlled, in situ release of proteins. Chapter 6 describes the 3D bioprinting of pHPMAlac-PEG/HAMA hydrogels in combination with a polycaprolactone (PCL)-based reinforcement for the development of cartilage composite constructs with relevant stiffness and high chondrogenic potential. In Chapter 7, we report about the ectopic implantation of pHPMAlac-PEG/HAMA hydrogels in small animal (murine) and large animal (equine) models, as well as the orthotopic implantation in a large animal (porcine) model, as a work up for the future orthotopic implantation in a highly challenging equine model. Importantly, pHPMAlac-PEG/HAMA hydrogels showed adequate biocompatibility in the tested species and locations. Nevertheless, variability in hydrogel stability and resistance between different implantation sites and among the different species highlights the need for further optimization before an orthotopic, long-term screening in horses is undertaken.